Project description:We present Nanopore-DamID, a method to simultaneously detect cytosine methylation and DNA-protein interactions from single molecules, via selective sequencing of adenine-labelled DNA. Assaying LaminB1 and CTCF binding with Nanopore-DamID, we identify escape from LAD-associated repression of hypomethylated promoters amidst generalised hypermethylation of LaminB1-associated regulatory elements. We detect novel CTCF binding sites in highly repetitive regions, and allele-specific CTCF binding to imprinted genes and the active X chromosome. Nanopore-DamID highlights the importance of DNA methylation to transcription factor activity.
Project description:The Oxford Nanopore (ONT) platform provides portable and rapid genome sequencing, and its ability to natively profile DNA methylation without complex sample processing is attractive for clinical sequencing. We recently demonstrated ONT shallow whole-genome sequencing to detect copy number alterations (CNA) from the circulating tumor DNA (ctDNA) of cancer patients. Here, we show that cell-type and cancer-specific methylation changes can also be detected, as well as cancer-associated fragmentation signatures. This feasibility study suggests that ONT shallow WGS could be a powerful tool for liquid biopsy, especially real-time medical applications.
Project description:Using a public reference data set of 82 unique entities, 382 nanopore-sequenced brain tumor samples were classified based on their methylation status through an ad hoc random forest algorithm. As a measure of confidence, score recalibration was performed and platform-specific thresholds were defined.
Project description:We used the nanopore Cas9 targeted sequencing (nCATS) strategy to specifically sequence 125 L1HS-containing loci in parallel and measure their DNA methylation levels using nanopore long-read sequencing. Each targeted locus is sequenced at high coverage (~45X) with unambiguously mapped reads spanning the entire L1 element, as well as its flanking sequences over several kilobases. The genome-wide profile of L1 methylation was also assessed by bs-ATLAS-seq in the same cell lines (E-MTAB-10895).
Project description:Probing epigenetic features on long molecules of DNA has tremendous potential to advance our understanding of the phased epigenome. In this study, we evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA using GpC methyltransferase to exogenously label open chromatin, coupled with nanopore sequencing technology. We performed nanopore sequencing of Nucleosome Occupancy and Methylome (nanoNOMe) on four human cell lines (GM12878, MCF-10A, MCF-7, MDA-MB-231), and demonstrate the ability to directly measure methylation and chromatin accessibility in genomic features such as structural variations and repetitive elements. The long single-molecule resolution allows footprinting of protein and nucleosome binding and determining the combinatorial promoter epigenetic state on individual molecules. Long-read sequencing makes it possible to robustly assign reads to haplotypes, enabling allele-specific epigenetic analysis across the genome. We use existing SNV data on GM12878 to present the first fully phased human Probing epigenetic features on long molecules of DNA has tremendous potential to advance our understanding of the phased epigenome. We evaluate CpG methylation and chromatin accessibility simultaneously on long strands of DNA using GpC methyltransferase to exogenously label open chromatin, coupled with nanopore sequencing technology. We performed nanopore sequencing of Nucleosome Occupancy and Methylome (nanoNOMe) on four human cell lines (GM12878, MCF-10A, MCF-7, MDA-MB-231), and demonstrate the ability to directly measure methylation and chromatin accessibility in genomic features such as structural variations and repetitive elements. The long single-molecule resolution allows footprinting of protein and nucleosome binding and determining the combinatorial promoter epigenetic state on individual molecules. Long-read sequencing makes it possible to robustly assign reads to haplotypes, enabling allele-specific epigenetic analysis across the genome. We use existing SNV data on GM12878 to present the first fully phased human epigenome, consisting of chromosome-level allele-specific profiles of CpG methylation and chromatin accessibility.mosome-level allele-specific profiles of CpG methylation and chromatin accessibility.
Project description:The Oxford Nanopore technology has a great potential for the analysis of genome methylation, including full-genome methylome profiling. However, there are certain issues while identifying methylation motif sequences caused by low sensitivity of the currently available motif enrichment algorithms. Here, we present Snapper, a new highly-sensitive approach to extract methylation motif sequences based on a greedy motif selection algorithm. Snapper has shown higher enrichment sensitivity compared with the MEME tool coupled with Tombo or Nanodisco instruments, which was demonstrated on H. pylori strain J99 studied earlier using the PacBio technology. In addition, we used Snapper to characterize the total methylome of a new H.pylori strain A45. The analysis revealed the presence of at least 4 methylation sites that have not been described for H. pylori earlier. We experimentally confirmed a new CCAG-specific methyltransferase and indirectly inferred a new CCAAK-specific methyltransferase.
Project description:Epigenetic characterization of cell-free DNA (cfDNA) is an emerging approach for detecting and characterizing diseases such as cancer. We developed a strategy using nanopore-based single-molecule sequencing to measure cfDNA methylomes. This approach generated up to 200 million reads for a single cfDNA sample from cancer patients, an order of magnitude improvement over existing nanopore sequencing methods. We developed a single-molecule classifier to determine whether individual reads originated from a tumor or immune cells. Leveraging methylomes of matched tumors and immune cells, we characterized cfDNA methylomes of cancer patients for longitudinal monitoring during treatment.